Generally, specific proliferation and differentiation factors are required for the proliferation and differentiation of hematopoietic cells. Such factors participate in the differentiation and proliferation of hematopoietic cells into various blood cells, such as erythrocytes, granulocytes, macrophages, eosinophils, platelets and lymphocytes (Yasusada Miura, "Blood Stem Cells," Chugai Igaku Sha, 1983). Of these factors, CSFs are known as factors which stimulate the proliferation and differentiation of granulocyte precursor cells and macrophage precursor cells. Among the CSF's are G-CSF which is specific to the formation of granulocytes, M-CSF which is specific to the formation of macrophages, GM-CSF which acts to form both granulocytes and macrophages and multi-CSF(IL-3) which stimulates pluripotent stem cells.
The above-mentioned CSFs, because of their biological activity, are thought to alleviate the leukopenia resulting from cancer chemotherapy and radiotherapy as a common drawback of these therapies. Clininal research on CSFs is conducted from this viewpoint.
The CSFs are also known to have activity to promote the function of leukocytes (Lopez, A. F. et al., J. Immunol., 131, 2983 (1983), Handam, E. et al., same, 122, 1134 (1979) and Vadas, M. A. et al., same, 130, 795 (1983)) and are therefore found effective as drugs for preventing and curing various infectious diseases.
Furthermore, the CSFs are found effective for curing myelogenous leukemia because of its differentiation inducing activity (Metcalf, D. et al., Int. J. Cancer, 30, 773 (1982)).
The activity of CSFs is found, for example, in the cultures of fetal cells, spleen cells, etc. in human urine and in the culture media of various incubated cell lines, and active fractions thereof are separated off and used. However, the CSF of any origin contains large quantities of analogous extraneous substances or the like derived from the starting material and is in itself low in concentration, so that these problems must be solved in preparing the CSF. In respect of homogeneity, yield, ease of operation, etc., therefore, methods still remain to be developed of commercially preparing CSFs as drugs.
We have already established "AGR-ON," a cell line derived from human leukemic T cells, which is capable of producing a large quantity of CSF constitutively in a homogeneous state (Unexamined Japanese Patent Publication SHO 59-169489). We have also developed a process for preparing a CSF from AGR-ON easily in a pure form and in a high yield and further clarified the structural and biochemical characteristics of the resulting CSF (Unexamined Japanese Patent Publication SHO 62-169799).
The CSF disclosed by the above invention is M-CSF, i.e., a glycoprotein which acts on normal bone marrow cells to promote the differentiation and proliferation of macrophages and which is characterized by the following physicochemical properties. The CSF is termed "AGR-ON.CSF."
a) Molecular weight
33,000 to 43,000 daltons as determined by SDS polyacrylamide gel electrophoresis under a non-reducing condition, and 23,000 to 40,000 daltons as determined by gel filtration under a non-reducing condition in the presence of SDS.
b) N-terminal amino acid sequence of the protein portion
The sequence of the following primary structural formula. ##STR1##